Flashcards in Proteomics Deck (30):
The study of the entire complement of proteins, how they're modified, when and where they're expressed, how they're involved in metabolic pathways and how they interact with one another
What are proteins made up of?
What is special about proteins?
They all have distinctive molecular weights
Give an example of an enzymatic catalysis protein
Give an example of a transport and storage protein
Give an example of a coordinated motion protein
Give an example of an immune protection protein
Give examples of proteins involved in the generation and transmission of nerve impulses
Rhodopsin and acetylcholine
Give an example of a protein involved in the control of growth and differentitation
Growth factor proteins
What are the two proteomics approaches?
Top down and bottom up
Briefly describe the bottom-up proteomics approach
1. Isolate proteins
2. Reduce and alkylate
4. Separate out peptides
5. Mass determine peptides and sequences
6. Informally collate all of the data to determine what the protein is and how much there is
Proteins are structurally complicated structures, true or false?
How can you break the four levels of protein structure?
By trypsin digest
Give an example of a reducing agent, how it works, and what the outcome is for proteins
Disrupts disulphide bonds to free thiol groups
Give an example of an alkylating agent, how it works, and what the outcome is for proteins
Maintains disrupted disulphide bonds by alkylating cysteine groups and thus preventing them from reforming disulphide bonds
Causes the formation of S-carbamidomethyl cysteine groups
What do digestive enzymes do?
Recognise specific amino acids and cleaves at this specific site only
What is the most commonly used digestive enzyme?
Where does trypsin cut?
At the C-terminus of lysine and arginine residues (except when either is followed by proline)
What sorts of peptides will be excluded from digestion analyses?
Very short and very long peptides
What do you need to do in order to maximise the liberation of tryptic peptides?
Need to disrupt the 3D structure of proteins
What is dithiothreitol (DTT) used for?
To reduce/disrupt disulphide bonds formed between cysteine groups
What is iodoacetamide (IAA) used for?
To alkylate cysteine groups to prevent the reformation of disulphide bonds
What is trypsin used for?
To generate MS amenable peptides
What do samples that are a mixture of peptides have a range of?
What is utilised to establish a separation gradient on HPLC?
The wide range of hydrophobicities
As peptides elute from LC column what are they subject to?
Vaporisation, ionisation and detection by the mass spectrometer
What are ionised peptides subject to?
Tandem mass spectrometry to determine amino acid sequence
What does LC stand for?
What is liquid chromatography similar to?